Discovery and functional characterization of a novel PilV variant in a mcr-1 -positive IncI2 plasmid

IncI2-type plasmids are major vehicles for the dissemination of mcr-1 . These plasmids encode a type IV secretion system (T4SS) that mediates conjugative transfer and a type IV pilus (T4P) that facilitates mating-pair formation. Shufflon-mediated diversity of tip adhesin PilV of T4P may contribute to the extensive dissemination of IncI2 plasmids. Herein, we identified a novel shufflon segment forming an active new PilV variant from an IncI2 plasmid, which was named PilV-E and was detected in 127 of 501 (25.35%) complete IncI2 plasmids in public databases. We further demonstrated that strains harboring the single PilV-E variant displayed a distinct functional profile, characterized by both robust biofilm formation and high Caco-2 adhesion. Compared with the Δ pilV -shufflon- rci strain, the PilV-E-carrying strain showed increased biofilm formation (OD ₅₉₀ from 1.50 to 2.60), increased Caco-2 adhesion (0.04% to 5.46%), and increased surface motility (1.20 to 1.87 cm/day). Among all variants, PilV-A' showed the strongest cell adhesion (6.00%), whereas PilV-B' showed the greatest motility (2.54 cm/day). Notably, PilV-E significantly restored conjugation frequency compared with the Δ pilV -shufflon- rci strain and displayed the highest and lowest frequencies when recipients were Escherichia coli (5.23 × 10 ⁻³ ) and Acinetobacter baumannii (4.46 × 10 ⁻⁸ ), respectively. All findings confirm that PilV-E serves as a novel and key tip adhesin of T4P and plays a crucial role in plasmid conjugation, indicating that the diversity of shufflon-mediated PilV variants has been underestimated and providing insight into the molecular basis for the extensive spread of IncI2 plasmids.